The present invention relates to use of α-cyano-4-hydroxycinnamic acid derivatives as a matrix in MALDI mass spectrometry of analytes.
Mass spectrometry (MS) is a common analytical technique used to sort ionized molecules in a sample by their mass-to-charge ratio. A challenge within the field of MS is that ionization can damage molecules within a sample, with organic molecules and biomolecules being particularly susceptible. The matrix-assisted laser desorption/ionization (MALDI) technique was developed in the 1980's as a way to ionize biomolecules and large organic molecules with reduced molecule fragmentation. The technique involves mixing the molecules of interest (analyte) with a matrix material and irradiating the combined sample with a laser; this irradiation releases charges from the matrix that ionize the analyte.
MALDI relies on the selection of a proper organic matrix molecule. Several criteria must be met in order for an organic molecule to be a useful MALDI matrix. Because most UV-MALDI mass spectrometers incorporate a pulsed laser, commonly either a nitrogen (337 nm) or frequency tripled Nd:YAG laser (355 nm), the matrix must contain a chromophore which allows for sufficient absorption at the chosen wavelength. This absorption is essential for electronic excitation of the molecule to occur. Secondly, the matrix must be weakly acidic, as this allows for the donation of a proton from the matrix to the analyte. While some matrices exhibit several acidic protons, it is commonly the most acidic proton in the excited or ion state which is transferred to the analyte during ionization. Another important consideration when determining a suitable matrix is the ability of the matrix to co-crystallize with the analyte. This co-crystallization between matrix and analyte is essential as it brings the analyte into the gas-phase upon desorption. Subsequently, the matrix helps create gas-phase ions of the analyte with minimal fragmentation. Typical analytes that are suitable for MALDI-MS include biomolecules (e.g., oligonucleotides such as DNA and RNA, proteins, peptides, sugars, lipids, medical substances, plant metabolites, etc.) and large organic molecules (e.g., polymers, dendrimers, catenanes, rotaxanes, and other macromolecules).
The analysis begins by mixing a large molar excess of the matrix with the analyte. The ratio of matrix to analyte, typically 500:1 to 5000:1, is often varied to achieve optimal signal-to-noise ratio. Various deposition methods for both the matrix and analyte have been thoroughly investigated, including the dried droplet, fast evaporation, and slow crystallization approaches. These methods all encompass a few crucial steps in the sample preparation. First, a drop of matrix solution, typically 1 μL, is placed onto the stainless steel MALDI target and allowed to dry. 1 μL of the analyte solution is then placed onto the dried matrix spot and is also allowed to air dry. Upon drying of the analyte solution, co-crystallization of the two compounds can occur to form inhomogeneous matrix-analyte crystals. These inhomogeneous crystals are then irradiated by the pulsed laser which results in desorption and ionization of the matrix and analyte. A dense plume of desorbed matrix and analyte is formed, from which ions are accelerated down the flight tube of the MS to be detected individually.
Common MALDI matrices include 2,3-dihydroxybenzoic acid (2,3-DHB), 2,4-dihydroxybenzoic acid (2,4-DHB), 2,5-dihydroxybenzoic acid (2,5-DHB), 2,6-dihydroxybenzoic acid (2,6-DHB), 3,4-dihydroxybenzoic acid (3,4-DHB), 3,5-dihydroxybenzoic acid (3,5-DHB), α-cyano-4-hydroxycinnamic acid (CHCA), ferulic acid (FA), and sinapic acid (SA), 2,4,6-trihydroxyacetophenone.
Although commonly referred to as the “gold standard” MALDI-MS matrix, MS performed using CHCA matrices commonly suffer from lack of signal reproducibility. Specifically, the analyte ion signals supported by CHCA lack the sensitivity required to effectively analyze many analytes.
Here, it was found that the introduction of heavy atoms (e.g., halogens such as fluoro, chloro, bromo, or iodo substitution) to the aromatic ring of CHCA, while retaining the 4-OH group, overcomes these disadvantages. In particular, the addition of halogens (i.e., F, Cl, Br, or I) to the aromatic ring of CHCA has drastically improved analyte ionization, ion signal reproducibility (demonstrated from relative standard deviation), and interferences from low-mass ions, relative to the non-substituted matrix.